IADR Abstract Archives
Up-take of Porphyromonas Gingivalis Membrane Molecules and PD-L1 Up-regulation
Objectives: Expression of PD-L1 on host cells modulates immune response. During inflammation this receptor is induced on epithelial cells and alters T-cell responses. It was demonstrated that the membrane fraction of P. gingivalis is able to induce PD-L1 up-regulation. After up-take of the bacterial molecules this effect is triggered by NOD1/NOD2 binding. Endocytosis is one potential mechanism for the up-take of substances into the cell and is a constitutive property of most eukaryotic cells. It allows non-specific internalization of nutrients in addition to receptor-mediated internalization of molecules. The aim of this study was to analyze the up-take of bacterial components by carcinoma cells that subsequently induce PD-L1 up-regulation.
Methods: Squamous cell carcinoma cells (SCC-25 ) were seeded (1 x 106 cells) and stimulated with P. gingivalis W83 total membrane (TM) fraction - obtained by mechanical lysis following ultracentrifugation of P. gingivalis W83. After 24h cells were harvested and prepared for Western blot. PD-L1 expression was investigated by immuno-staining. For endocytosis analysis TM was coupled to the pHrodo™ GreenSTP ester. Up-take of the fluorescent TM was analyzed by flow cytometry.
Results: Induction of PD-L1 protein expression was detected in dose- and time-dependent manner after stimulation with P. gingivalis W83 membrane fraction. After stimulation with fluorochrome-ester labelled TM the number of fluorescent cells increased from 8.3% (±3) after 20 min to 55.5% (±13) after 48 h (n = 3) (p < 0.01), indicating that the majority of cells internalized TM via endocytosis.
Conclusions: We provide evidence that TM of P. gingivalis up-regulates the immune-regulatory receptor PD-L1 in oral carcinoma cells and that TM enters the cells via endocytotic up-take.
Acknowledgements: This study was supported by the von-Behring-Röntgen Stiftung and the Deutsche Forschungsgemeinschaft (DFG).
Methods: Squamous cell carcinoma cells (SCC-25 ) were seeded (1 x 106 cells) and stimulated with P. gingivalis W83 total membrane (TM) fraction - obtained by mechanical lysis following ultracentrifugation of P. gingivalis W83. After 24h cells were harvested and prepared for Western blot. PD-L1 expression was investigated by immuno-staining. For endocytosis analysis TM was coupled to the pHrodo™ GreenSTP ester. Up-take of the fluorescent TM was analyzed by flow cytometry.
Results: Induction of PD-L1 protein expression was detected in dose- and time-dependent manner after stimulation with P. gingivalis W83 membrane fraction. After stimulation with fluorochrome-ester labelled TM the number of fluorescent cells increased from 8.3% (±3) after 20 min to 55.5% (±13) after 48 h (n = 3) (p < 0.01), indicating that the majority of cells internalized TM via endocytosis.
Conclusions: We provide evidence that TM of P. gingivalis up-regulates the immune-regulatory receptor PD-L1 in oral carcinoma cells and that TM enters the cells via endocytotic up-take.
Acknowledgements: This study was supported by the von-Behring-Röntgen Stiftung and the Deutsche Forschungsgemeinschaft (DFG).